For eradication of cancer cells in the body, cellular immunity, particularly cytotoxic T cells (cytotoxic T-lymphocytes, Cytotoxic T-cells, hereinafter to be referred to as CTLs) mainly play an important role. CTLs are produced by differentiation and proliferation of precursor T cells that have recognized a complex formed by an antigen peptide derived from a cancer antigen protein (cancer antigen peptide) and an MHC class I molecule, and attacks cancer cells. MHC in human is called human leukocyte-type antigen (HLA), and HLA subtypes such as HLA-A, B, Cw, F and G are known.
A cancer antigen peptide is produced through degradation (processing) of a cancer antigen protein synthesized in cancer cells, i.e., protein denaturation by reduction of a sulfur-sulfur covalent bond, degradation by proteosome or protease, cleavage into an optimum length by a trimming enzyme in the endoplasmic reticulum of the protein. The cancer antigen protein generally consists of 8-12 amino acid residues.
In cancer immunotherapy, activation of helper T cells is also important for activating other T cells including CTLs. In general, an antigen protein is degraded by intracellular lysosome, and a part of the peptide fragments, each of which is a peptide consisting of about 13-17 amino acid residues, binds as an antigen peptide to MHC class II molecule and is presented to helper T cell-TCR.CD3 complex to activate helper T cells. In human, HLA subtypes such as HLA-DR, DQ and DP are known.
As an antigen of a cancer vaccine, a cancer antigen protein itself or an antigen peptide derived from a cancer antigen protein is mainly used (see Non Patent Literature 1). Since a cancer vaccine using a protein generally contains various cancer antigen peptides, it can simultaneously induce a plurality of CTLs and helper T cells. However, the cancer protein vaccine possesses problems in stable supply and quality control. On the other hand, a cancer vaccine using a peptide can be conveniently produced or quality-controlled, but is mainly constituted by a single MHC class I-presented peptide antigen. Thus, it has been pointed out in recent years that efficient induction of CTLs requires further improvement (see Non Patent Literature 2).
One of the solutions for such problems is a multivalent antigen peptide-presenting peptide cancer vaccine. As such peptide cancer vaccines, a cocktail vaccine containing a mixture of a plurality of peptide antigens to be presented by MHC class I and class II, a long chain peptide vaccine containing peptide antigens to be presented by MHC class I and class II which are bound by an amide bond, and the like have been reported (see Non Patent Literature 2). In the case of a cocktail vaccine, however, since each peptide antigen composed of various amino acids shows various physical properties, the development of an optimal formulation capable of efficiently inducing CTLs corresponding thereto is often problematic. In the case of a long chain peptide vaccine, its production sometimes has problems similar to those of production of a protein. Furthermore, since the peptide antigens to be presented by class I and class II are bonded via a peptide spacer in a long chain peptide vaccine, it is difficult to control and predict the cleavage sites by intracellular enzyme. In the meantime, a peptide dimer wherein two peptide monomers are mutually bonded by a disulfide bond has been reported (see Patent Literature 1). Different from cocktail vaccine, a homodimer has two single peptides being bonded, and therefore, they have single physical property and can be produced conveniently. On the other hand, cancer antigen peptides are required to contain cysteine in their amino acid sequences, and therefore, applicable peptides are limited.
The process of presentation of a cancer antigen peptide on MHC class I involves a plurality of peptidases. Of such peptidases, Endoplasmic reticulum aminopeptidase 1 (hereinafter to be referred to as ERAP1) is one of the trimming enzymes in the endoplasmic reticulum (hereinafter to be referred to as ER), and has been reported to recognize a particular antigen peptide sequence and peptide length, and cleaves a cancer antigen peptide precursor from the N-terminal to control the length to be optimal for binding to MHC class I (see Non Patent Literature 3). However, there is no report to date on a conjugate vaccine using the trimming function of ERAP1. Moreover, although ERAP1 has been reported to convert a precursor peptide to a cancer antigen peptide by cleaving dicysteine from the N-terminal, it has been unclear whether the trimming is affected when an amino acid sequence containing cysteine is introduced to the N-terminal (see non-patent documents 3-6).